Stroke is a potentially devastating neurological disorder with high morbidity and mortality. Thrombolytic agents such as tissue plasminogen activator (t-PA) are currently the only drugs available to reverse acute ischemic stroke, but reestablishment of circulation may paradoxically initiate a reperfusion injury. Therefore, there is great interest in developing treatments to limit reperfusion injury. Recent studies with experimental stroke models, mouse genetics, and selective peptide inhibitors and activators have implicated PKC4 in stroke-reperfusion injury. In particular, PKC4 expressed in neutrophils appears essential for stroke- reperfusion injury. Understanding the molecular and cellular actions of PKC4 in reperfusion injury requires the identification of the unique targets of PKC4 in signaling pathways activated by ischemia and reperfusion. A novel chemical-genetics approach has been developed to identify immediate substrates of kinases. Based on this approach, we have generated a PKC4-analog sensitive mutant (PKC4-as) enzyme that can utilize specific ATP analogs that are not accepted by other kinases, and recently we succeeded in generating a knock-in mouse expressing PKC4-as. With these reagents, we have the unique opportunity to identify direct substrates of PKC4 in response to stroke-reperfusion injury. This project will first characterize the PKC4-as mice. In the second specific aim, we will use this novel chemical-genetics approach to label and identify PKC4 substrates in neutrophils. We will then validate these isolated substrates in the third specific aim. This work will reveal PKC4 signaling pathways activated during cerebral ischemia and reperfusion in neutrophils, and should facilitate the development of PKC4-related therapeutic strategies. Ischemic stroke can potentially be reversed by tissue plasminogen activator (t-PA), but the restoration of blood flow to ischemic areas can paradoxically produce reperfusion injury. Much evidence supports a key role of protein kinase C delta in reperfusion injury. This proposal is to identify the targets of protein kinase C delta, and the information obtained will facilitate the development of new therapies to limit stroke- reperfusion injury.